OPEN ACCESS International Journal of Molecular Sciences...hair growth [2].Platelet-derived growth...

Int. J. Mol. Sci. 2015, 16, 4379-4391; doi:10.3390/ijms16034379

International Journal of

Molecular Sciences ISSN 1422-0067

www.mdpi.com/journal/ijms

Article

Hair-Growth-Promoting Effect of Conditioned Medium of High Integrin α6 and Low CD 71 (α6

bri/CD71dim) Positive Keratinocyte Cells

Chong Hyun Won 1, Yun-Mi Jeong 2, Sangjin Kang 2, Tae-Sung Koo 3, So-Hyun Park 4,

Ki-Young Park 5,Young-Kwan Sung 6 and Jong-Hyuk Sung 7,*

1 Department of Dermatology, Asan Medical Center, University of Ulsan College of Medicine, Seoul

138-736, Korea; E-Mail: [email protected] 2 Department of Applied Bioscience, CHA University, Seoul 135-081, Korea;

E-Mails: [email protected] (Y.M.J.); [email protected] (S.K.) 3 Graduate School of New Drug Discovery and Development, Chungnam National University,

Daejeon 305-764, Korea; E-Mail: [email protected] 4 Coway Cosmetics R&D Center, Seoul 153-792, Korea; E-Mail: [email protected] 5 Asan Institute for Life Sciences, Seoul 138-736, Korea; E-Mail: [email protected] 6 Department of Immunology, School of Medicine, Kyungpook National University, Daegu 700-422,

Korea; E-Mail: [email protected] 7 College of Pharmacy, Yonsei University, Incheon 406-840, Korea

* Author to whom correspondence should be addressed; E-Mail: [email protected];

Tel.: +82-32-749-4506; Fax: +82-2-486-7831.

Academic Editor: Bing Yan

Received: 22 October 2014 / Accepted: 11 February 2015 / Published: 19 February 2015

Abstract: Keratinocyte stem/progenitor cells (KSCs) reside in the bulge region of the hair

follicles and may be involved in hair growth. Hair follicle dermal papilla cells (HFDPCs)

and outer root sheath (ORS) cells were treated with conditioned medium (CM) of KSCs.

Moreover, the effects of KSC-CM on hair growth were examined ex vivo and in vivo.

A human growth factor chip array and RT-PCR were employed to identify enriched proteins

in KSC-CM as compared with CM from keratinocytes. KSC-CM significantly increased the

proliferation of HFDPCs and ORS cells, and increased the S-phase of the cell cycle

in HFDPCs. KSC-CM led to the phosphorylation of ATK and ERK1/2 in both cell types.

After subcutaneous injection of KSC-CM in C3H/HeN mice, a significant increase in hair

growth and increased proliferation of hair matrix keratinocytes ex vivo was observed.

OPEN ACCESS

Int. J. Mol. Sci. 2015, 16 4380

We identified six proteins enriched in KSC-CM (amphiregulin, insulin-like growth factor

binding protein-2, insulin-like growth factor binding protein-5, granulocyte

macrophage-colony stimulating factor, Platelet-derived growth factor-AA, and vascular

endothelial growth factor). A growth-factor cocktail that contains these six recombinant

growth factors significantly increased the proliferation of HFDPCs and ORS cells and

enhanced the hair growth of mouse models. These results collectively indicate that KSC-CM has

the potential to increase hair growth via the proliferative capacity of HFDPCs and ORS cells.

Keywords: conditioned media; hair growth; keratinocyte stem cells; paracrine effect

1. Introduction

The hair follicle (HF) is a unique characteristic of mammals; its structure undergoes cyclic

transformation from stages of rapid growth (anagen) to apoptosis-driven regression (catagen) via

an interspersed period of relative quiescence (telogen). The cycling and regeneration of each HF depends

on specialized mesenchymal dermal papilla cells and proliferating matrix cells located at the base of the

follicle, and which are mediated by several molecules that control epithelial morphogenesis and growth.

Several growth factors reportedly stimulate hair growth in ex vivo and animal models.

For example, controlled release of vascular endothelial growth factor (VEGF) promotes the hair growth

of the murine HF [1], and VEGF-mediated angiogenesis improves follicle vascularization and increases

hair growth [2]. Platelet-derived growth factor (PDGF) isoforms reportedly induce and maintain the

anagen phase of the murine HF and promote hair regeneration in mice [3]. Hepatocyte growth factor

(HGF) and insulin-like growth factor (IGF) have also demonstrated the ability to up-regulate HF growth

in various systems, such as, murine HF morphogenesis and cycling [4–7].

Keratinocyte stem/progenitor cells (KSCs) are known to reside in mostly two locations, the bulge

region of the hair follicle and in non-random distributions within the basal layer of inter-follicular

epithelium. KSCs, with a long-term proliferative possibility and a high short-term colony forming

efficiency, express the integrin α6 strongly but weakly express a proliferation-associated cell surface

marker, named transferrin receptor (CD71) (α6bri/CD71dim) [8,9]. KSCs continuously undergo self-

renewal and generate transit-amplifying cells that rapidly divide to supply skin with new epithelial cells

in the epidermal regeneration process. Moreover, KSCs play a critical role of in hair regeneration. For

example, Kamimura et al. reported that HFs are composed of diverse cells of multiple origins and that

the vast majority of reconstituted follicles appeared to be derived from KSCs [10]. In addition, Taylor

et al. demonstrated that KSCs in a bulge give rise to HF cells as well as to upper follicular

cells [11]. The KSC niche was defined by demonstrating that the bulge area contains the majority of

infrequently cycling, label-retaining cells (i.e., KSCs), which can respond to the anagen phase inducing

signals from dermal papilla cells to regenerate hair follicle [12]. From previous study [13] and in this

work, we defined high integrin α6 and low CD 71 (α6bri/CD71dim) positive keratinocyte cells as KSCs, as

these subsets of keratinocytes have some characteristics of stem cells in the skin.

Secretion of growth factors and activation of neighboring cells are the origins of the mechanism of

action of stem cells, and we have previously demonstrated that secretomes of adipose-derived stem cells

Int. J. Mol. Sci. 2015, 16 4381

promote hair growth in both HF organ culture and in animal studies [14]. Likewise, secretomes from

KSCs may play a key role in hair regeneration via a paracrine mechanism in the HF. Therefore, in our

present study we investigated: (1) whether KSC-conditioned medium (KSC-CM) promotes hair growth;

and (2) which factor(s) secreted from KSCs mediate(s) hair-growth promotion. We studied the hair-

growth promotion effects of KSC-CM on human HF organ culture and on a C3H/HeN mouse model. We

then investigated the proliferative effect of KSC-CM on both human hair follicle dermal papilla cells

(HFDPCs) and outer root sheath (ORS) cells.

2. Results

2.1. Effects of Keratinocyte Stem/Progenitor Cells Conditioned Medium (KSC-CM) on Hair Follicle

Dermal Papilla Cells (HFDPCs)

Because hair cycle changes are influenced by rapid remodeling of both epithelial and dermal

components [2], we investigated the proliferation of both HFDPCs and ORS cells. First, concentrated

KSC-CM (1- and 5-fold) treatment significantly enhanced the proliferation of cultured HFDPCs

(Figure 1A). To clarify the underlying mechanism of increased proliferation of HFDPCs, we analyzed

the expression of signaling proteins related to cell proliferation using western blot analysis in HFDPCs.

The level of phosphorylated AKT was significantly increased after concentrated (5-fold) KSC-CM

treatment (Figure 1B). The expression of phosphorylated ERK1/2 was also increased with KSC-CM

incubation (Figure 1B).

Because KSC-CM increased the proliferation of HFDPCs, cell cycle analysis of HFDPCs was

performed in the presence of KSC-CM to determine whether the cell cycle was affected. Compared with

the control group, incubation with concentrated KSC-CM for 24 h and 48 h decreased the length of the

gap phase 1 (G1) of the cell cycle, while the proportion of the DNA synthesis phase (S) was increased

(Figure 1C,D). This result indicates that KSC-CM has proliferative effects on HFDPCs by increasing

the S phase fraction of the cell cycle.

Figure 1. Cont.

Int. J. Mol. Sci. 2015, 16 4382

Figure 1. Effects of keratinocyte stem/progenitor cells conditioned medium (KSC-CM) on

the proliferation of hair follicle dermal papilla cells (HFDPCs). (A) Cell proliferation was

measured 48 h after concentrated KSC-CM treatment; (B) Western blot analysis against

phospho-AKT and phospho-ERK1/2 showed that these pathways are activated by KSC-CM;

(C,D) Cell cycle analysis of HFDPCs was performed by flow cytometry, and we found that

the fraction of S phase cells was increased following 24 h (C) and 48 h (D) of concentrated

KSC-CM treatment. ** p < 0.01.

2.2. Effects of KSC-CM on Outer Root Sheath Cells

We investigated the proliferation of ORS cells and determined that treatment with 1- and 5-fold

concentrated KSC-CM significantly enhanced the proliferation of cultured ORS cells (Figure 2A).

The level of phosphorylated AKT was increased 5-fold after concentrated KSC-CM treatment

(Figure 2B). In addition, the expression of phosphorylated ERK1/2 was significantly increased with

KSC-CM incubation (Figure 2B). However, the cell cycle in KSC-CM treated ORS cells was not

affected (data not shown).

Figure 2. Effects of keratinocyte stem/progenitor cells conditioned medium (KSC-CM) on

outer root sheath (ORS) cell proliferation. (A) Cell proliferation was measured 48 h

after concentrated KSC-CM treatment; (B) Western blot analysis against phospho-AKT

and phospho-ERK1/2 showed that these pathways are activated by KSC-CM. * p < 0.05;

** p < 0.01.

Int. J. Mol. Sci. 2015, 16 4383

2.3. Hair-Growth-Promoting Effects of KSC-CM in a C3H/HeN Mouse Model

After topical injection of KSC-CM (Figure 3A) into the back of C3H/HeN mice, the conversion of

telogen to anagen was induced earlier than in the controls (Figure 3A). The area close to the injection

sites in the mice became darker in color after 10 days, thus indicating that HFs were in the anagen

phase of the hair cycle. However, the injection sites of the controls retained their original white color.

These findings indicate that locally injected KSC-CM might influence hair growth in vivo. In addition,

the hair weight measured 14 days after concentrated KSC-CM treatment indicated that KSC-CM

treatment significantly increased hair growth in the mice (Figure 3B). These findings indicate that

KSC-CM can induce an earlier conversion of the hair cycle because the media stimulated hair growth in

the murine model.

Figure 3. The promoting effects of keratinocyte stem/progenitor cells conditioned

medium (KSC-CM) on hair growth in mice. Telogen-matched, seven-week-old C3H/NeH

mice were shaved and subcutaneously injected three times with concentrated KSC-CM.

(A) Photographs were taken two weeks after the injections; (B) The new hair was shaved,

and the hair weight was measured. ** p < 0.01.

2.4. KSC-CM Increased Proliferation of Hair Matrix Keratinocytes Ex Vivo

The potential effect of KSC-CM on hair shaft elongation was investigated in isolated human anagen

hairs. After the addition of KSC-CM (0%, 2.5% and 10%) to William’s E medium, Ki67-positive cells

were stained green in ex vivo hair organ cultures. After two days of organ culture, the fluorescence

intensity in the 2.5% KSC-CM-treated group increased by 40% compared with the control group treated

Int. J. Mol. Sci. 2015, 16 4384

with William’s E media alone (Figure 4). These results indicate that KSC-CM may stimulate hair growth

by inducing the proliferation of follicular cells.

Figure 4. Effects of conditioned medium of keratinocyte stem/progenitor cells (KSC-CM)

on ex vivo organ culture of human hair follicles. Isolated human anagen hairs were treated

with KSC-CM (0%, 2.5% and 10%) in William’s E medium for two days. The Ki67-positive

cells (green) were significantly increased by KSC-CM treatment; cells were counter-stained

with 4',6-diamidino-2-phenylindole (DAPI).

2.5. Identification of Proteins Involved in the Hair-Growth Promotion by KSC-CM

Because a paracrine effect is likely to be important for KSC-mediated hair regeneration, we examined

the difference in secretion between keratinocytes and KSCs using a growth factor chip array [15–18].

The secretion of 41 growth factors was detected in conditioned medium obtained after a three-day culture

with keratinocytes or KSCs. The secretion of amphiregulin (AREG), insulin-like growth factor binding

protein-2 (IGFBP2), insulin-like growth factor binding protein-5 (IGFBP5), granulocyte macrophage-

colony stimulating factor (GM-CSF), PDGF-AA, and VEGF was significantly increased in KSC-CM

(Figure 5A). Consistent with the results seen in Figure 5A, RT-PCR analysis showed that KSCs showed

significantly higher mRNA levels of AREG, IGFBP2, IGFBP5, GM-CSF, PDGF-AA, and VEGF than did

keratinocytes (Figure 5B).

Int. J. Mol. Sci. 2015, 16 4385

Figure 5. Altered protein secretion and mRNA expression in keratinocyte stem/progenitor

cells (KSCs) and keratinocytes. (A) The growth-factor chip array showed that secretion of

AREG, IGFBP2, IGFBP5, GM-CSF, PDGF-AA, and VEGF were significantly increased;

(B) In addition, the mRNA expression of these growth factors is highly expressed in KSCs.

2.6. Hair-Growth-Promotion Effect of the Growth-Factor Complex

Because six growth factors are highly expressed in KSCs compared with keratinocytes, we investigated

the hair-growth-promotion effect of the growth-factor cocktail GFC, which is a mixture of a 1 ng/mL

concentration of these six recombinant growth factors. Figure 6A shows that GFC induced the

conversion of the HF from telogen to anagen phase in C3H/HeN mice and also increased the hair weight

(Figure 6A). In addition, GFC treatment (a mixture of a 1 ng/mL or 10 ng/mL concentration of the six

recombinant growth factors) significantly induced the proliferation of HFDPCs (Figure 6B) and of ORS

cells (Figure 6C). Collectively, these results indicate that these growth factors might be responsible for

the hair-growth-promotion effect of KSC-CM.

Figure 6. Cont.

Int. J. Mol. Sci. 2015, 16 4386

Figure 6. Effect of the growth-factor cocktail (GFC) on hair growth. (A) Seven-week-old

C3H/NeH mice were shaved and subcutaneously injected three times with GFC. The hair

weight was significantly increased by GFC treatment. In addition; GFC treatment significantly

increased the proliferation of hair follicle dermal papilla cells (HFDPCs) (B) and outer root

sheath (ORS) (C) cells. ** p < 0.01.

3. Discussion

Because KSCs may have a key role in hair regeneration via a paracrine mechanism, in the present

study we investigated whether KSC-CM could promote hair growth as well as which factors in KSC-CM

might mediate this process. We found that concentrated KSC-CM significantly increased the

proliferation of HFDPCs and ORS cells, in part, through the phosphorylation of AKT and ERK1/2.

Concentrated KSC-CM treatment accelerated hair growth in C3H/HeN mice and increased the

proliferative fraction of Ki67-positive hair matrix keratinocytes in human HFs. We identified six secreted

growth factors that were enriched in KSC-CM and found that a cocktail of these proteins

(1 ng/mL concentration of each protein) accelerated the hair growth in a mouse model as well as

HFDPCs and ORS cells.

In a preliminary study, we co-cultured keratinocytes and HFDPC cells in a trans-well system, but

found that keratinocytes did not increase the proliferation of DPCs (data not shown). Therefore,

we identified the six growth factors by comparing the secretomes of KSCs and keratinocytes using

a growth-factor chip array. KSC-CM contains proliferative stimulators including angiogenic factors such

as AREG, IGFBP2, IGFBP5, GM-CSF, PDGF-AA, and VEGF. We found that the combination of these

factors stimulates hair growth. RT-PCR analysis also confirmed that KSCs express high basal levels of

AREG, IGFBP2, IGFBP6, GM-CSF, PDGF-AA, and VEGF mRNAs compared with keratinocytes

(Figure 5B). However, it is unlikely that only these factors are involved in the hair growth of KSCs.

Because of the limitations of the assay system we used in this study, we could measure only 41 growth

factors in the conditioned medium. Therefore, there may be additional factors which accelerate the hair

growth in KSC-CM. Further studies will be needed to identify the key paracrine factors. We also tested

whether the addition of a single growth factor could increase the proliferation of HFDPCs, and found

that no single factor significantly increased the proliferation in our preliminary study. Therefore,

it is reasonable to conclude that the growth factors in KSC-CM collectively promote hair growth.

The positive roles of growth factors on hair-growth stimulation are well-known. IGF-1 is essential for

normal hair growth and development, and may also be important in regulating the hair-growth

Int. J. Mol. Sci. 2015, 16 4387

cycle [19]. In many culture systems IGF-1 actions are modulated by the IGFBPs, which are also

reportedly involved in controlling hair growth [7,20]. In mice, PDGF isoforms markedly promote hair

regeneration by regulating the anagen phase of the murine HF [3]. Controlled release of VEGF

increases hair growth in the murine HF [1], and VEGF-mediated angiogenesis also improves follicle

vascularization as well [2]. However, the functional roles of AREG and GM-CSF on hair growth have

not yet been reported.

4. Experimental Section

4.1. Cell Cultures

HFDPCs were obtained from PromoCell (Heidelberg, Germany) and grown in Follicle Dermal

Papilla Cell Growth Medium with Supplement Mix (PromoCell) at 37 °C in 5% CO2. ORS cells and

keratinocytes were cultured as previously described [21], and were grown in EpiLife (GIBCO, Invitrogen,

Carlsbad, CA, USA) at 37 °C in 5% CO2. KSCs were obtained from CELLnTEC (Bern, Switzerland)

and were grown in Keratinocyte Growth Medium 2 with Supplement Mix (PromoCell) at 37 °C in 5%

CO2. KSCs were characterized by flow cytometry (FACS) analysis using antibodies to integrin α6 and

CD 71 as described in a previous study [13].

4.2. Preparation and Concentration of KSC-CM

Concentrated KSC-CM was prepared according to the method of Kim et al. [22,23], although with

some modifications. Briefly, KSCs became 100% confluent, and the medium was replaced

with serum-free medium. After 72 h, conditioned medium was collected, centrifuged at 1800 rpm

for 10 min, and filtered through a 0.22-μm syringe filter. The filtrate was then centrifuged

in 3 kDa molecular-weight-cut-off Vivaspin (Sartorius Stedim Biotech GmbH, Goettingen, Germany)

and concentrated.

4.3. Proliferation Assay

HFDPCs or ORS cells were seeded at a density of 5000 cells/cm2 in 48-well plates. After 24 h of

incubation, the culture medium was replaced by new medium containing various concentrations of

concentrated KSC-CM, and the cells were then incubated for 72 h in order to proliferate. A cell

proliferation assay was then performed using a Cell-Counting Kit-8 (CCK-8, Dojindo Laboratories,

Kumamoto, Japan). CCK-8 solution (150 μL) was added, and each well was incubated for 2 h.

Following incubation, the absorbance was measured at 450 nm using an enzyme-linked immunosorbent

assay (ELISA) reader (TECAN, Grodig, Austria).

4.4. FACS Analysis

FACS analysis was performed as previously described [22]. After serum starvation for 24 h,

cells were incubated using supplement-free media or concentrated KSC-CM. After 48 h, cells were then

harvested, washed twice with PBS, permeabilized with 70% ethanol, and finally stained with 0.2 mg/mL

RNase A and 50 μg/mL propidium iodide. The cells were analyzed for the DNA cycle using a flow

cytometer (Becton-Dickinson, San Jose, CA, USA).

Int. J. Mol. Sci. 2015, 16 4388

4.5. Western Blotting

Protein samples were prepared in RIPA buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM

EDTA, 1% Triton-X 100, 1% SDS, 50 mM NaF, 1 mM Na3VO4, 5 mM dithiothreitol, 1 mg/mL leupeptin,

and 1 mM PMSF). Samples were separated on 12% SDS-polyacrylamide gel, transferred to PVDF

membranes, and blocked with 5% dried milk in PBS containing 0.4% Tween-20. The blots were

incubated with the appropriate primary antibodies that recognize AKT, phospho-specific AKT

(Ser473; Cell Signaling, Danvers, MA, USA), ERK1/2 (Cell Signaling), phospho-specific ERK1/2

(Cell Signaling), and actin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Membrane-bound

primary antibodies were detected using secondary antibodies conjugated with horseradish peroxidase

and Immobilon™ western chemiluminescent HRP reagent (Millipore, Bedford, MA, USA) and exposed

to X-ray film (Agfa, Mortsel, Belgium).

4.6. Animal Studies

Protocols for the animal studies were approved by the Institutional Animal Care and Use Committee

of the Seoul National University (Seoul, Korea). Female C3H/HeN mice (6 weeks old and weighing 20–

25 g) were purchased from Orient Bio, Inc. (Seongnam, Korea). The mice were kept in a clean room

(Animal Center for Pharmaceutical Research, College of Pharmacy, Seoul National University) at a

temperature between 20–23 °C, with 12 h light (07:00–19:00) and dark (19:00–07:00) cycles, and with

a relative humidity of 50% ± 5%. The mice were housed in metabolic cages (Tecniplast, Varese, Italy)

in filtered, pathogen-free air and with both food (Agribrands Purina, Pyeongtaek, Korea) and water

available ad libitum. Hair growth studies were conducted in seven-week-old C3H/HeN mice; HFs were

synchronously matched in the telogen stage. The dorsal side of each mouse was shaved using a clipper

and electric shaver with special care taken in order to avoid damaging the bare skin. Three subcutaneous

injections of 0.1% BSA-PBS (control), growth-factor cocktail (GFC), and concentrated KSC-CM were

then made in the dorsal skin of each mouse at two-day intervals, and any darkening of the skin was

monitored. After two weeks, the re-grown dorsal hair was shaved and its weight was measured.

4.7. Ex Vivo Human HF Organ Culture Study

Human occipital scalp HFs were isolated from volunteers who had given informed consent, and the

HFs were cultured in vitro, as described previously [24]. Briefly, dissected HFs were cut into small

pieces, approximately 2 mm in length from the bottom of the dermal papilla, and were cultured in

William’s E medium (Gibco BRL, Gaithersburg, MD, USA) with 10 ng/mL hydrocortisone, 10 ng/mL

insulin, 2 mM L-glutamine, and 100 U/mL penicillin at 37 °C in a 5% CO2 atmosphere. Anagen HFs

were cultured from three different volunteers. KSC-CM (0%, 2.5% and 10%) was added to the

basal William’s E medium. HFs cultured in William’s E media were used for a negative control.

After culturing for two days, the HF organs were then harvested and were ultimately stained with

anti-Ki-67 (1:100; Becton Dickinson, Franklin Lakes, NJ, USA) and DAPI.

Int. J. Mol. Sci. 2015, 16 4389

4.8. Human Growth Factor Chip Array

A human growth factor chip array kit was purchased from R&D systems (Minneapolis, MN, USA).

Briefly, membranes were placed in an 8-well tissue culture tray and were incubated with 2 mL of

1× blocking buffer at room temperature for 1 h. The membrane was then incubated overnight at 4 °C

with 1 mL of concentrated control or concentrated KSC-CM (total 200 μg of protein). After decanting

the samples, 1 mL of diluted detection antibody cocktail A was incubated with a membrane for 2 h at

room temperature. After incubation with diluted streptavidin-HRP at room temperature for 30 min,

a signal was detected using a chemiluminescent substrate system (Immobilon western reagent), and the

signal was quantified using a densitometer (Bio-Rad Laboratories, Hercules, CA, USA). The background

intensity was subtracted for the analysis. The data were normalized to the positive control values

expressed in the membrane.

4.9. Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) Analysis

Total RNA was extracted using TRIzol reagent (Invitrogen) followed by reverse transcription

using a cDNA synthesis kit (Promega, Madison, WI, USA). cDNA was synthesized from 1 µg of

total RNA using 200 U of reverse transcriptase and 20 pM oligo-dT. The following oligonucleotides

were used as primers: VEGF (5'-TAC CTC CAC CAT GCC AAG T-3' and

5'-TGC ATT CAC ATT TGT TGT GC-3'); AREG (5'-AAG CGT GAA CCA TTT TCT GG-3' and

5'-AGC CAG GTA TTT GTG GTT CG-3'); GM-CSF (5'- ACT GCT GCT GAG ATG AAT GA-3' and

5'-AGG GCA GTG CTG CTT GTA GT-3'); IGFBP-2 (5'-CCC TCA AGT CGG GTA TGA AG-3' and

5'-ACC TGG TCC AGT TCC TGT TG-3'); IGFBP-5 (5'-GAA TCC AGG CAC CTC TAC CA-3' and

5'-GGT AGA AGC CTC GAT GGT CA-3'); PDGF-AA (5'-CAA GAC CAG GAC GGT CAT TT-3' and

5'-CCT GAC GTA TTC CAC CTT GG-3'); and the internal control GADPH (5'-CGA GAT CCC TCC

AAA ATC AA-3' and 5'-TGT GGT CAT GAG TCC TTC CA-3'). PCRs were performed in a final

volume of 20 µL of reaction mix that contained 2 µL of the RT reaction mixture, 15 mM MgCl2,

1.25 mM dNTPs, 20 pM of each primer, and 0.5 U of Taq polymerase (Promega). Thermal cycling

consisted of an initial denaturation at 94 °C for 5 min, amplification for 35 cycles (94 °C for 30 s,

56 °C for 30 s, and 72 °C for 30 s), and termination by a final extension at 72 °C for 5 min. The GAPDH

mRNA level was used for sample standardization. After electrophoresis on 1.5% agarose gel, each band

was quantified using a densitometer (Bio-Rad Laboratories).

4.10. Statistics

Differences among treatments were assessed by an analysis of variance (ANOVA) and followed by

Dunnett’s test. p-values of <0.05 were regarded as significant.

5. Conclusions

In this study, we have found that high integrin α6 and low CD 71 (α6bri/CD71dim) positive

keratinocyte cells increases the proliferation of HFDPCs and ORS cells. High integrin α6 and

low CD 71 (α6bri/CD71dim) positive keratinocyte cells treatment accelerates hair growth in C3H/HeN

mice and increases the number of Ki67-positive hair matrix keratinocytes. Thus, high integrin α6 and

Int. J. Mol. Sci. 2015, 16 4390

low CD 71 (α6bri/CD71dim) positive keratinocyte cells or a mixture of recombinant growth factors can be

used for hair-growth stimulation.

Acknowledgments

The manuscript has been professionally edited by Kilian Perrem, Boston BioEdit.

Conflicts of Interest

The authors declare no conflict of interest.

References

1. Ozeki, M.; Tabata, Y. Promoted growth of murine hair follicles through controlled release of

vascular endothelial growth factor. Biomaterials 2002, 23, 2367–2373.

2. Yano, K.; Brown, L.F.; Detmar, M. Control of hair growth and follicle size by VEGF-mediated

angiogenesis. J. Clin. Investig. 2001, 107, 409–417.

3. Tomita, Y.; Akiyama, M.; Shimizu, H. PDGF isoforms induce and maintain anagen phase of

murine hair follicles. J. Dermatol. Sci. 2006, 43, 105–115.

4. Jindo, T.; Tsuboi, R.; Takamori, K.; Ogawa, H. Local injection of hepatocyte growth factor/scatter

factor (HGF/SF) alters cyclic growth of murine hair follicles. J. Investig. Dermatol. 1998, 110,

338–342.

5. Lindner, G.; Menrad, A.; Gherardi, E.; Merlino, G.; Welker, P.; Handjiski, B.; Roloff, B.; Paus, R.

Involvement of hepatocyte growth factor/scatter factor and met receptor signaling in hair follicle

morphogenesis and cycling. FASEB J. 2000, 14, 319–332.

6. Su, H.Y.; Hickford, J.G.; Bickerstaffe, R.; Palmer, B.R. Insulin-like growth factor 1 and hair

growth. Dermatol. Online J. 1999, 5, 1

7. Weger, N.; Schlake, T. IGF-I signalling controls the hair growth cycle and the differentiation of

hair shafts. J. Investig. Dermatol. 2005, 125, 873–882.

8. Kaur, P. Interfollicular epidermal stem cells: Identification, challenges, potential. J. Investig. Dermatol.

2006, 126, 1450–1458.

9. Webb, A.; Li, A.; Kaur, P. Location and phenotype of human adult keratinocyte stem cells of the

skin. Differentiation 2004, 72, 387–395.

10. Kamimura, J.; Lee, D.; Baden, H.P.; Brissette, J.; Dotto, G.P. Primary mouse keratinocyte cultures

contain hair follicle progenitor cells with multiple differentiation potential. J. Investing. Dermatol.

1997, 109, 534–540.

11. Taylor, G.; Lehrer, M.S.; Jensen, P.J.; Sun, T.T.; Lavker, R.M. Involvement of follicular stem cells

in forming not only the follicle but also the epidermis. Cell 2000, 102, 451–461.

12. Tumbar, T.; Guasch, G.; Greco, V.; Blanpain, C.; Lowry, W.E.; Rendl, M.; Fuchs, E. Defining the

epithelial stem cell niche in skin. Science 2004, 303, 359–363.

13. Sung, S.H.; Park, S.H.; Song, S.Y.; Lee, S.J.; Lee, H.W.; Kim, S.H.; A Lee, M.; Yoon, I.S.;

Kim, D.D.; Kang, S.; et al. Epidermal regeneration by ENT-16α, 17-dihydroxy-kauran-19-oic acid

isolated from Siegesbeckia pubescens. Cell Prolif. 2011, 44, 527–536.

Int. J. Mol. Sci. 2015, 16 4391

14. Won, C.H.; Yoo, H.G.; Kwon, O.S.; Sung, M.Y.; Kang, Y.J.; Chung, J.H.; Park, B.S.; Sung, J.H.;

Kim, W.S.; Kim, K.H.; et al. Hair growth promoting effects of adipose tissue-derived stem cells.

J. Dermatol. Sci. 2010, 57, 134–137.

15. Kim, W.S.; Park, B.S.; Sung, J.H. Protective role of adipose-derived stem cells and their soluble

factors in photoaging. Arch. Dermatol. Res. 2009, 301, 329–336.

16. Park, B.S.; Jang, K.A.; Sung, J.H.; Park, J.S.; Kwon, Y.H.; Kim, K.J.; Kim, W.S. Adipose-derived

stem cells and their secretory factors as a promising therapy for skin aging. Dermatol. Surg. 2008,

34, 1323–1326.

17. Song, S.Y.; Chung, H.M.; Sung, J.H. The pivotal role of VEGF in adipose-derived-stem-cell-

mediated regeneration. Expert Opin. Biol. Ther. 2010, 10, 1529–1537.

18. Lee, E.Y.; Xia, Y.; Kim, W.S.; Kim, M.H.; Kim, T.H.; Kim, K.J.; Park, B.S.; Sung, J.H.

Hypoxia-enhanced wound-healing function of adipose-derived stem cells: Increase in stem cell

proliferation and up-regulation of VEGF and bFGF. Wound Repair Regen. 2009, 17, 540–547.

19. Batch, J.A.; Mercuri, F.A.; Werther, G.A. Identification and localization of insulin-like growth

factor-binding protein (IGFBP) messenger RNAs in human hair follicle dermal papilla.

J. Investig. Dermatol. 1996, 106, 471–475.

20. Park, B.S.; Kim, W.S.; Choi, J.S.; Kim, H.K.; Won, J.H.; Ohkubo, F.; Fukuoka, H. Hair growth

stimulated by conditioned medium of adipose-derived stem cells is enhanced by hypoxia: Evidence

of increased growth factor secretion. Biomed. Res. 2010, 31, 27–34.

21. Kwack, M.H.; Shin, S.H.; Kim, S.R.; Im, S.U.; Han, I.S.; Kim, M.K.; Kim, J.C.; Sung, Y.K.

L-Ascorbic acid 2-phosphate promotes elongation of hair shafts via the secretion of insulin-like

growth factor-1 from dermal papilla cells through phosphatidylinositol 3-kinase. Br. J. Dermatol.

2009, 160, 1157–1162.

22. Kim, W.S.; Park, B.S.; Park, S.H.; Kim, H.K.; Sung, J.H. Antiwrinkle effect of adipose-derived

stem cell: Activation of dermal fibroblast by secretory factors. J. Dermatol. Sci. 2009, 53, 96–102.

23. Kim, W.S.; Park, B.S.; Kim, H.K.; Park, J.S.; Kim, K.J.; Choi, J.S.; Chung, S.J.; Kim, D.D.;

Sung, J.H. Evidence supporting antioxidant action of adipose-derived stem cells: Protection of

human dermal fibroblasts from oxidative stress. J. Dermatol. Sci. 2008, 49, 133–142.

24. Krugluger, W.; Rohrbacher, W.; Laciak, K.; Moser, K.; Moser, C.; Hugeneck, J. Reorganization

of hair follicles in human skin organ culture induced by cultured human follicle-derived cells.

Exp. Dermatol. 2005, 14, 580–585.

© 2015 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article

distributed under the terms and conditions of the Creative Commons Attribution license

(http://creativecommons.org/licenses/by/4.0/).

OPEN ACCESS International Journal of Molecular Sciences...hair growth [2].Platelet-derived growth...

Documents

Transcript of OPEN ACCESS International Journal of Molecular Sciences...hair growth [2].Platelet-derived growth...